Nitric oxide (NO) is an important inhibitory neurotransmitter for the gastrointestinal smooth muscles. As a neurotransmitter, NO plays a key role in peristalsis and physiological relaxation of the sphincters and thus facilitates transport of food through the gastrointestinal tract. Loss of NO-related neurotransmission has been shown to cause diseases causing difficulty in swallowing, gastric stasis, dyspepsia, intestinal stasis and constipation. Defective NO neurotransmission is also implicated in diabetic gastroparesis and functional disorders of the gut such as irritable bowel syndrome and spasm of biliary and pancreatic sphincters. Nitric oxide is uniquely different from other classical neurotransmitters. Unlike the classical neurotransmitters, NO is a highly diffusible gas that is produced de novo on demand from Ca-CaM dependent activation of nNOS. However, regulation of NO generation and nitrergic neurotransmission is not well understood. We have recently shown that isolated nitrergic varicosities from mice gut contain inactive and active pools of nNOS. Dynamic regulation of the catalytically active nNOS1 is responsible for regulation of nitrergic neurotransmission. Overall purpose of the proposed studies is to extend our studies of regulation of catalytically active nNOS in the nitrergic varicosities and determine how nNOS is transported to the membrane, attached there and regulated to produce NO. We will use this information to identify abnormities in the steps that may impair nitrergic neurotransmission. These studies will help define pathophysiology of impaired nitrergic neurotransmission that have no anatomical evidence of loss or damage to nitrergic nerves. There are four specific aims: 1) To examine the role of PIN/LC8 and myosin Va in targeting nNOS to varicosity membrane and to investigate nitrergic neurotransmission in mice lacking myosin Va. 2) To examine the role of PSD proteins in membrane association of nNOS1 dimer and to investigate nitrergic neurotransmission with depalmitoylation of PSD and in mice lacking PSD 95. 3) To investigate the clustering of nNOS-PSD complex with N-type calcium channels and the enzymes that dephosphorylate or phosphorylate nNOS and to investigate the effects of inhibitors of these enzymes on nitrergic neurotransmission. 4) To investigate the mechanism of reduced inhibitory neurotransmission in an animal model of diabetes mellitus (NOD mice).